Cloning and Sequence Analysis of Actin Gene from Rehmannia glutinosa

[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.

cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua

[Objective] This study aims to conduct cloning and sequence analysis of ADH gene in D. Antiqua. [Method] Full-length cDNA of ADH gene in D. antiqua was cloned by using RACE technology （GenBank access number： JQ666006）. Analysis of the homology, characteristics and functional domains of ADH sequence and the phy- Iogenetic relationship to other dipteran ADH were conducted. [Result] The full length of ADH cDNA is 1 088 bp containing a 771 bp of ORF, encoding 256 amino acids, with a calculated relative molecular weight of 30.80 kDa and a theoretical isoelectric point of 8.22. The deduced amino acid sequence shares the highest homology with Glossina morsitans morsitans based on homological analysis and phylogenetic analysis. [Conclusion] This study provides basis for further research of ADH gene.

Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

PCR Based Detection and Phylogenetic Analysis of Fowl Adenovirus Strains Isolated from 2019 Epidemic from Punjab and Sindh, Pakistan

Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.

Repetitive sequence analysis and karyotyping reveal different genome evolution and speciation of diploid and tetraploid Tripsacum dactyloides

In the subtribe Maydeae, Tripsacum and Zea are closely related genera. Tripsacum is a horticultural crop widely used as pasture forage. Previous studies suggested that Tripsacum might play an important role in maize origin and evolution. However, our understanding of the genomics and the evolution of Tripsacum remains limited. In this study, two diploids,T. dactyloides var. meridionale(2n = 36, MR) and T. dactyloides(2n = 36, DD), and one tetraploid,T. dactyloides(2n = 72, DL) were sequenced by low-coverage genome sequencing followed by graph-based cluster analysis. The results showed that 63.23%, 59.20%, and 61.57% of the respective genome of MR, DD, and DL were repetitive DNA sequence. The proportions of different repetitive sequences varied greatly among the three species. Fluorescence in situ hybridization(FISH) analysis of mitotic metaphase chromosomes with satellite repeats as the probes showed that the FISH signal patterns of DL were more similar to that of DD than to that of MR. Comparative analysis of the repeats also showed that DL shared more common repeat families with DD than with MR. Phylogenetic analysis of internal transcribed spacer region sequences further supported the evolutionary relationship among the three species. Repetitive sequences comparison showed that Tripsacum shared more repeat families with Zea than with Coix and Sorghum. Our study sheds new light on the genomics of Tripsacum and differential speciation in the Poaceae family.

利用Brn1基因分析嘴突脐孢种(Exserohilum rostratum内变异研究

对形态变异特征较大的嘴突脐孢种Brn1基因核苷酸序列系统发育进行了分析。从系统发育树中可以看出,供试菌株与突脐孢属的其它菌种聚类在同一个分支上,与弯孢属和离蠕孢属的菌种明显区分开,形成了一个独立演化群。利用DNADIST程序计算遗传距离矩阵分析所有供试嘴突脐孢种菌株间的最小遗传距离值和最大遗传距离值分别为0.0078和0.0197,供试嘴突脐孢种菌株间的遗传距离值变化较大,但该种菌株间距离值小于系统树中种内最大遗传距离值0.0242。这一结果表明嘴突脐孢种内不同菌株间的变异属于种内变异。Brn1基因核苷酸序列分析可以用于嘴突脐孢种鉴定。

Nucleotide sequence characterization and phylogenetic analysis of hantaviruses isolated in Shandong Province,China

Background China is the most severe endemic area of hemorrhagic fever with renal syndrome （HFRS） in the world with 30 000-50 000 cases reported annually, which accounts for more than 90% of total number of cases worldwide. The incidence rate of the syndrome in Shandong Province is one of the highest in China, which has ever reached 50 per 100 000 persons per year. However, the molecular characteristics of hantaviruses （HV） epidemic in Shandong Province remain unclear. Therefore it is useful to clarify nucleotide sequence and phylogenetic characteristics of HV isolated in Shandong Province in order to provide better advices to control and prevent HFRS. Methods RNAs were extracted from sera of clinically diagnosed patients and positive rodent lungs that were detected by indirect immunofluorescent assay （IFA）. Partial M segments of HV were amplified from the RNAs with reverse transcription nested polymerase chain reactions （nested PCR） using hantavirus genotype specific primers. The nested PCR products were sequenced and compared with those from previously epidemic isolates in Shandong and with other representative HV sequences from GenBank. Phylogenetic tree analyses were performed based on the sequences of the M genes. Results Thirty-four HV isolates in Shandong showed 67.1%-100% nucleotide identities. The nucleotide homologies among 6 Hantaan viruses （HTNV） isolates in Shandong were 78.1%-98.7%, while the homologies among 28 Seoul virus （SEOV） isolates in Shandong were 93.7%-100%. There were at least 3 subtypes HTNV （H2, H5, H9） and 2 subtypes SEOV （S2, S3） in Shandong Province. Conclusions In Shandong Province, the homologies of HTNV were lower and there were no predominant subtypes, while the homologies of SEOV were higher and S3 was the predominant subtype. The homologies of SEOV from rodents were higher than those from patients. The distribution of subtypes in Shandong was similar to that of the adjoining provinces. Phylogenetic analyses of the sequences showed geographic clustering of HV in Shandong.

Complete chloroplast genome sequences of Schisandra chinensis:genome structure,comparative analysis,and phylogenetic relationship of basal angiosperms

Dear Editor, Schisandra chinensis （Turcz.） Baill. belongs to family Schisandraceae. Its fruit called ＂Wu Wei Zi＂ in Chinese is a well-known medicinal material, which is used to treat chronic cough and dyspnea, nocturnal emission, enuresis, etc. （National Pharmacopoeia Committee, 2015）. Except for S. chinensis, many species of Schisandraceae, such as S. sphenanthera, S. lancifolia and S. rubriflora, are used as the original olants of folk medicines.

Determination and Analysis of Mitochondrial ND2 Gene Sequence of Anas platyrhynchos

[Objective] The study was to analyze the phylogenesis of Anas platyrhynchos. [Method] Complete sequence of mitochondrial ND2 gene of 4 Anas platyrhynchos was determined by direct DNA sequencing based on PCR products. Combined with ND2 gene sequences of the Anas Linnaeus accessed in GenBank, phylogenetic tree was constructed by Neighbor-joining and maximum parsimony methods. [Result] The ND2 gene sequences of 4 Anas platyrhynchos were identical(1 041 bp in length; the nucleotide contents of A, G, T, and C were 28.91%, 13.35%, 20.75% and 36.98% respectively; A+T content approximated to that of C+G). Sequences of ND2 gene of mallard were same as spotbill duck, and had high homology with others. The phylogenetic trees indicated mallard and spotbilled duck were close in genetic relationship, both shared a haplotype; then Philippine duck, green-winged teal and northern pintail fell into branch ''A". [Conclusion] The domestic duck may be domesticated from mallard and spotbilled duck.

Phylogenetic Analysis of Cystatin

[Objective] The molecular weight,isoelectric point,signal peptide,domain and other properties of the encoding protein of the known cystatin genes were analyzed.[Method] Cystatin genes were searched in NCBI and the related amino acids sequences were downloaded.SMART software was used to predict the domain.SingalP program was used to search signal peptide.TMHMM program was used to search and predict the transmembrane domain.CLUSTAL W program was used to make multiple sequence alignment.Using MEGA3.1 software,...

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge,few reports on Demodex studied at the molecular level are available at present.In this study our group,for the first time,cloned,sequenced and analyzed the chitin synthase(CHS) gene fragments of Demodex folliculorum,Demodex brevis,and Demodex canis(three isolates from each species) from Xi'an China,by designing specific primers based on the only partial sequence of the CHS gene of D.canis from Japan,retrieved from GenBank.Results show that amplification was successful only in three D.canis isolates and one D.brevis isolate out of the nine Demodex isolates.The obtained fragments were sequenced to be 339 bp for D.canis and 338 bp for D.brevis.The CHS gene sequence similarities between the three Xi'an D.canis isolates and one Japanese D.canis isolate ranged from 99.7% to 100.0%,and those between four D.canis isolates and one D.brevis isolate were 99.1%-99.4%.Phylogenetic trees based on maximum parsimony(MP) and maximum likelihood(ML) methods shared the same clusters,according with the traditional classification.Two open reading frames(ORFs) were identified in each CHS gene sequenced,and their corresponding amino acid sequences were located at the catalytic domain.The relatively conserved sequences could be deduced to be a CHS class A gene,which is associated with chitin synthesis in the integument of Demodex mites.

Classification and Identification of Marine Penicillium Species Based on ITS Sequences of rDNA

[ Objective ] The aim of this study is to identify 6 marine fungi species by analyzing ITS nucleotide sequence, which had been primarily identified as penicillium based on morphological characteristics. [ Method ] The ITS regions of these species were cloned by molecule biology method and phylogenetic analyzed using ClustalX1.83 software. [ Result] The ITS regions of these species were sequenced, and phylogenetic analysis between the yield sequences and the ITS sequences assessed in GenBank showed that the 6 strains all belonged to penicillium. [ Condusion] The present study suggests ITS sequence analysis could not be used as an only proof, but it is a very useful supplementary tool for the classification and identification of marine peniciUium combined with morphological characteristics.

The complete mitochondrial genome of Oreta fuscopurpurea(Lepidoptera: Drepanidae) and its phylogenetic implications

In this study, the first complete mitochondrial genome（mitogenome） of lepidopteran family Drepanidae（superfamily Drepanoidea） was reported with the notes about its phylogenetic implications. The Oreta fuscopurpurea mitogenome is 15,564 bp in length, including 13 protein-coding genes, two ribosomal RNAs（lrRNA and srRNA）, 22 transfer RNAs and a non-coding control region（AT-rich region）, with a 79.6% A＋T content. The gene orientation and arrangement of the mitogenome are the same as other sequenced lepidopterans. All protein-coding genes usually start with the common ATN codon except for COI gene, which used CGA as the initial codon; eight PCGs use a typical stop codon of TAA, whereas the remaining PCGs use incomplete stop codon of T or TA. All tRNAs have the typical clover-leaf structure with the exception of t RNA^（Ser）（AGN）. The lrRNA and sr RNA genes are 1,409 bp and 778 bp in size respectively, with the former harboring one（TA）_（13） microsatellite-like repeat and an 17 bp insertion. The 20 intergenic spacers totaling of 184 bp and 8 overlapping sequences totaling of 25 bp are scattered throughout the whole mitogenome. The 526 bp AT-rich region contains some structures characteristic of lepidopterans, such as the motif ATAGA preceded by an 19 bp poly-T stretch, a tRNA-like and a sterm-loop structures. Phylogenetic analysis of the Oreta fuscopurpurea with other 47 insect species covering 20 lepidopteran families were conducted based on the sequence data of the 13 mitogenomic protein coding genes with maximum likelihood（ML） and Bayesian inference（BI） methods, and the results showed distinctly that the superfamily Drepanoidea was sister to the clade of（Bombycoidea, Lasiocampoidea） ＋（Noctuoidea, Geometroidea）.

Molecular analysis and anticancer properties of two identified isolates,Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2(ATCC) cell line

Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sub>50 value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.

Analysis of Mitochondrial DNA from the Ancient Tombs of Turfan

MtDNA was successfully extracted from ten individual bones (femurs) in the tombs of ancient Jushi in Turfan basin, dated back to the year about 3 000-2 500 years ago. By means of four overlapping primers, we got nucleotide sequence of the 218bp length. Ancient mtDNA was analyzed by the sequencing of hypervariable region Ⅰ of the mtDNA control region. The result shows that 9 haplotypes with 24 polymorphic sites were obtained. The phylogenetic analysis indicated that Mongolians and Altai are the population genetically closest to the Jushi groups and Jushi mtDNA pool being an admixture of eastern Asian and European lineages. So our preliminary data imply that an ancient mingling of Euro-Asian population had existed in Turfan basin prior to the early Iron Age.

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis:isolation and phylogenetic identification of the parasite

Background:Echinococcus multilocularis causes alveolar echinococcosis(AE)and is widely prevalent in Qinghai Province,China,where a number of different species have been identified as hosts.However,limited information is available on the Qinghai vole(Lasiopodomys fuscus),which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E.multilocularis.Thus,L.fuscus could contribute to the endemicity of AE in the area.Methods:Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E.multilocularis infection via anatomical examination.Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1.PCR-amplified segments were sequenced for a phylogenetic analysis.E.multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities.Results:Seventeen of the 50 Qinghai voles had E.multilocularis-infection-like vesicles in their livers.Eleven out of the 17 Qinghai voles presented E.multilocularis infection,which was detected by PCR and sequencing.The phylogenetic analysis showed that all 11 positive samples belonged to the E.multilocularis Asian genotype.A morphological identification and phylogenetic analysis of the E.multilocularis-infected Qinghai voles confirmed that all captured animals were L.fuscus.Conclusions:L.fuscus can be infected with E.multilocularis and plays a potential role in the life cycle and epidemiology of E.multilocularis in the Qinghai-Tibetan Plateau of China.

Origin and evolution of alginate-c5-mannuronan-epimerase gene based on transcriptomic analysis of brown algae

The coding product of alginate-c5-mannuronan-epimerase gene （algG gene） can catalyze the conversion of mannuronate to guluronate and determine the M/G ratio of alginate. Most of the current knowledge about genes involved in the alginate biosynthesis comes from bacterial systems. In this article, based on some algal and bacterial algG genes registered on GenBank and EMBL databases, we predicted 94 algG genes open reading frame （ORF） sequences of brown algae from the 1 000 Plant Transcriptome Sequencing Project （OneKP）. By method of transcriptomic sequence analysis, gene structure and gene localization analysis, multiple sequence alignment and phylogenetic tree construction, we studied the algal algG gene family characteristics, the structure modeling and conserved motifs of AlgG protein, the origin of alginate biosyn-thesis and the variation incidents that might have happened during evolution in algae. Although there are different members in the algal algG gene family, almost all of them harbor the conserved epimerase region. Based on the phylogenetic analysis of algG genes, we proposed that brown algae acquired the alginate bio-synthesis pathway from an ancient bacterium by horizontal gene transfer （HGT）. Afterwards, followed by duplications, chromosome disorder, mutation or recombination during evolution, brown algal algG genes were divided into different types.

Genetic Diversity of Chinese Soybean mosaic virus Strains and Their Relationships with Other Plant Potyviruses Based on P3 Gene Sequences

Soybean mosaic virus （SMV）, a member of the genus Potyvirus, is a major pathogen of soybean plants in China, and 16 SMV strains have been identified nationwide based on a former detailed SMV classification system. As the P3 gene is thought to be involved in viral replication, systemic infection, pathogenicity, and overcoming resistance, knowledge of the P3 gene sequences of SMV and other potyviruses would be useful in efforts to know the genetic relationships among them and control the disease. P3 gene sequences were obtained from representative isolates of the above-mentioned 16 SMV strains and were compared with other SMV strains and 16 Potyvirus species from the National Center for Biotechnology GenBank database. The P3 genes from the 16 SMV isolates are composed of 1041 nucleotides, encoding 347 amino acids, and share 90.7-100% nucleotide （NT） sequence identities and 95.1-100% amino acid （AA） sequence identities. The P3 coding regions of the 16 SMV isolates share high identities （92.4-98.9% NT and 96.0-100% AA） with the reported Korean isolates, followed by the USA isolates （88.5-97.9% NT and 91.4-98.6% AA）, and share low identities （80.5-85.2% NT and 82.1-84.7% AA） with the reported HZ 1 and P isolates from Pinellia ternata. The sequence identities of the P3 genes between SMV and the 16 potyviruses varied from 44.4 to 81.9% in the NT sequences and from 21.4 to 85.3% in the AA sequences, respectively. Among them, SMV was closely related to Watermelon mosaic virus （WMV）, with 76.0-81.9% NT and 77.5-85.3% AA identities. In addition, the SMV isolates and potyvirus species were clustered into six distinct groups. All the SMV strains isolated from soybean were clustered in Group I, and the remaining species were clustered in other groups. A multiple sequence alignment analysis of the C-terminal regions indicated that the P3 genes within a species were highly conserved, whereas those among species were relatively variable.

Cloning,Expression and Bioinformatic Analysis of Dorsal Gene in Delia antique

[ Objective ] The aim was to explore the relationship between the Dorsal and diapause development of Delia araiqua. [ Method ] The full-length cDNA of Dorsal in D. antiqua was cloned through RACE. The similarity among deduced amino acid sequence of Dorsal cDNA and the Dorsals of other 14 insect species were compared, and the phylogenetic analysis of these Dorsals was conducted. The expression level of Dorsal gene in winter-, summer- and non-diapausing pupae was analyzed. [ Result] The full-length of Dorsal cDNA sequence, 2 412 bp, was obtained with ORF 1974 bp, which coded 657 amino acids with predicted Mw 72.9 kDa and PI 8.5. The result of similarity comparison indicated that the DaDorsal was most related to those of Drosophlia melanogaster and Drosophlia pseudoobscura. The result of semi-quantitative RT-PCR analysis showed the expression level of Dorsal gene increased in characteristic duration of winter-, summer- and non- diapansing pupae, especially at the late diapanse, which might imply its relationship to D. antiqua diapause and development. [ Conclusion] The study lays the foundation for further study on gene function of Dorsal in insect diapause and development.